Gene therapy is an approach to treating diseases based on the expression of genes toward a therapeutic goal. Gene therapy has been discussed in the context of treating diseases although it also has a potential for disease prevention.
A basic challenge in gene therapy is to develop approaches for delivering genetic material to the appropriate cells of a patient in a way that is specific, efficient and safe. This problem of “drug delivery,” where the gene is a drug, is particularly challenging. If genes are appropriately delivered they can potentially lead to a cure. A primary focus of gene therapy is based on strategies for delivering genes.
Gene therapy promises to be a singular advance in the treatment of both acquired and genetic diseases at the most fundamental levels of pathology. Specifically, the development of gene transfer methods into the heart is attractive given that coronary artery disease is the leading cause of morbidity and mortality in the United States. Despite advances in the prevention and treatment of this disorder there remains a large population of patients who are not optimal candidates for percutaneous or surgical revascularization, usually because of severe distal vessel disease or previous failed revascularization procedures. Coronary collateral development is an important adaptive response of the ischemic heart in this situation, but often the collateral circulation is inadequate and results in severe angina pectoris despite maximal medical therapy. A new strategy to treat these often disabled patients involves the local delivery of vascular cytokines to induce new blood vessel growth (neoangiogenesis) in the ischemic myocardium. It has been recognized that gene therapy could play a major role in neovascularizarion approaches.
A variety of techniques has been developed to transfer genes into the heart. They have principally involved adenovirus vectors, which can be injected directly or intravenously, and plasmid DNA vectors injected directly into the heart tissue. The first reports of successful non-viral in vivo gene delivery to the heart used direct injection of plasmid DNA vectors (H. Lin, M. S. Parmacek, G. Morle, S. Bolling and J. M. Leiden. Circulation 82:2217-2221, 1990; G. Acsadi, S. S. Jiao, A. Jani, D. Duke, P. Williams, W. Chong and J. A. Wolff, New Biologist 3:71-81, 1991). High levels of β-galactosidase reporter gene expression were measured several days after injection of plasmid DNA solutions. Expression appeared to be highly localized to the site of injection. β-Galactosidase expression in the heart was not stable, apparently as the result of a host immune response against the expressing cells. Adenoviral vectors have been used extensively for gene transfer into cardiac muscle. Barr et al., found transduction levels of 10-32% after intracoronary installation. However, expression was also found in endothelial cells and the presence of the viral genome was detected in other organs (E. Barr, J. Carroll, A. M. Kalynych, S. K. Tripathy, K. Kozarsky, J. M. Wilson and J. M. Leiden. Gene Therapy 1:51-58, 1994). Many of these gene therapy studies were aimed at transducing vascular endothelial cells to prevent restenosis following angioplasty. The injection of adenoviral vectors into the portal or systemic circulatory systems leads to high levels of foreign gene expression in several organs (liver, lung, etc.) that is transient. As has been observed after adenovirus transduction of transgenes into other organs, expression in the heart is also transient. Immune responses directed against the viral coat proteins, proteins expressed from the viral genome, and the expressed transgene all contribute to the rapid elimination of transduced cells. Adenoviral transduction of infarcted heart tissue is less efficient than normal tissue. This could be a problem for viral gene therapy approaches for ischemic heart disease.
Until recently, the direct injection of plasmid DNA into the heart has mainly been used to benefit basic researchers investigating transcriptional regulation of cardiac specific genes. Isner and co-workers have pioneered the in vivo delivery of genes that result in neovascularization of ischemic muscle. In a breakthrough gene therapy study, they demonstrated significant formation of new vessels, enhanced distal flow, and clinical benefit in patients with ischemic limbs following injection of plasmid DNA expressing the human vascular endothelial growth factor (hVEGF) gene. This same hVEGF-expressing plasmid has recently been injected into ischemic heart tissue in humans. Preliminary results are very promising, with significant reduction in reported angina, and improved Rentrop score in 5 of 5 patients.
In vivo transfection of plasmid DNA complexed with liposomes after direct injection into heart muscle resulted in localized expression of reporter genes. While highly effective in vitro, liposome-complexed plasmid DNA particles generally have been of limited success in vivo. Other methods have involved the systemic delivery of adenoviral vectors or liposome-plasmid DNA complexes (e.g., injection into the tail vein of mice). Following systemic delivery, gene transfer into liver and lung is much more efficient than into heart, making this strategy unattractive for human cardiac gene therapy.
Vascular endothelial growth factor (VEGF) and various isoforms of fibroblast growth factor (FGF) are mitogens of endothelial and vascular smooth muscle growth in vitro and have been shown to induce neoangiogensis and ameliorate ischemia in animal models of vascular occlusion. In the rabbit hindlimb ischemia model, angiogenesis and increased collateralization have been demonstrated following intravenous, intra-arterial, and intramuscular injections of recombinant human VEGF. Similar effects of VEGF have been demonstrated in the coronary circulation. Banai and colleagues demonstrated that direct intracoronary injections of hVEGF (45 μg/d, 5 d/week×4 weeks) into an occluded coronary artery increased capillary density and improved coronary blood flow in a dog coronary occlusion model. Unfortunately, systemic arterial injections of VEGF did not improve collateral blood flow in the same model. Fibroblast growth factor likely has similar effects. Basic fibroblast growth factor has been shown to increase collateral blood flow in rat and rabbit hindlimb ischemia models. Repeated intracoronary and intra-arterial injections of basic fibroblast growth factor increased collateral circulation in the canine chronic coronary occlusion model. However, intravenous infusions of basic FGF protein are not efficacious in improving collateral coronary circulation in this model. While both VEGF or FGF induce neoangiogenesis and ameliorate ischemia in animal models of vascular occlusion, this strategy is clearly limited by the need for repetitive direct intra-arterial or intramuscular injections of the recombinant protein. In addition, the recombinant protein can cause significant systemic side-effects when administered in effective doses. An alternative strategy involves the use of sustained-release polymer to deliver cytokines locally to ischemic myocardium, but surgical implantation of these polymers is required. The difficulty and expense associated with large-scale recombinant protein manufacture is another limitation for potential clinical use of this strategy.
Local delivery of VEGF or FGF to ischemic muscle has also been achieved by gene transfer of complimentary deoxyribonucleic acid (cDNA) for these proteins via arterial infusion or direct needle injection of plasmid DNA. Direct intramuscular injection and percutaneous intraarterial injection of a human VEGF plasmid DNA expression vector induces collateral vessel development in the rabbit hindlimb ischemia model. Using the same model, it was demonstrated that intra-arterial injection of plasmids expressing either of three isoforms of human VEGF (hVEGF121, hVEGF165, and hVEGF189) are equally efficacious in inducing collateral vessel growth.
Using a porcine chronic coronary occlusion model, it was shown that single coronary injections of a replication-defective adenovirus vector expressing fibroblast growth factor-5 (FGF-5) resulted in improved regional myocardial blood flow and histologic evidence of increased capillary number. Stress-induced regional contractile dysfunction was documented by echocardiography after coronary occlusions in this study. Importantly, this pacing-induced regional wall motion abnormality was completely normalized within two weeks after intracoronary FGF-5 gene transfer and the amelioration of stress-induced myocardial ischemia persisted for at least 12 weeks. Again using the porcine model chronic coronary occlusion model, Mack et al. showed similar amelioration of pacing-induced myocardial ischemia and improvement in blood flow after multiple direct intramyocardial injections of a replication-defective adenovirus expressing hVEGF121.
Despite these limitations, the feasibility of these approaches to treating human vascular ischemic diseases has recently been demonstrated in two preliminary studies. Jeffrey Isner's group at St. Elizabeth's Medical Center in Boston recently reported encouraging preliminary results following intramuscular injection of naked plasmid DNA encoding hVEGF165 into the ischemic limb of patients suffering from severe peripherial vascular disease. Lower extremity perfusion (as measured by the ankle-brachial index) was improved in 7 of 10 treated patients.
In addition, improvement was noted in 4 of 7 patients with non-healing ischemic ulcers. Side effects were limited to lower extremity edema in 6 of the 10 treated patients. Neoangiogenesis in ischemic myocardium also showed clinical promise. Schumacher et al. injected recombinant basic fibroblast growth factor into myocardium surrounding the anastomosis of the internal mammary graft to the left anterior descending coronary artery in 20 patients undergoing coronary artery bypass surgery. Digital subtraction angiography demonstrated new capillary growth in the area of the injection.